Closing the Gap for Electronic Short‐Circuiting: Photosystem I Mixed Monolayers Enable Improved Anisotropic Electron Flow in Biophotovoltaic Devices

Abstract Well‐defined assemblies of photosynthetic protein complexes are required for an optimal performance of semi‐artificial energy conversion devices, capable of providing unidirectional electron flow when light‐harvesting proteins are interfaced with electrode surfaces. We present mixed photosystem I (PSI) monolayers constituted of native cyanobacterial PSI trimers in combination with isolated PSI monomers from the same organism. The resulting compact arrangement ensures a high density of photoactive protein complexes per unit area, providing the basis to effectively minimize short‐circuiting processes that typically limit the performance of PSI‐based bioelectrodes. The PSI film is further interfaced with redox polymers for optimal electron transfer, enabling highly efficient light‐induced photocurrent generation. Coupling of the photocathode with a [NiFeSe]‐hydrogenase confirms the possibility to realize light‐induced H2 evolution.

The Au electrode substrates were prepared using Si(100) wafers (Wacker) coated sequentially with titanium (as an adhesion layer) and gold by vapor deposition in a metal vaporization setup. Before use, the substrates were cleaned using Piranha solution (concentrated H2SO4 mixed with 30 wt.% H2O2 in a 3 to 1 volumetric ratio). Caution! Piranha solution is a powerful oxidizing agent and highly corrosive. Handle with extreme care.

Electrode Modification and Characterization
For deposition of thin P-Os films prior to monolayer transfer, the Au substrates were incubated overnight in a solution consisting of a mixture of P-Os (0.122 µg µL −1 ) and PEGDGE (0.5 µg mL −1 ) as cross-linker. The surface was then incubated for 30 min in a 50 mM Tris-HCl solution to induce polymer collapse. [1] Finally, the modified substrate was rinsed with water to remove any loosely adsorbed redox polymer.
The surface coverage of electrochemically active redox centers (ΓOs) was estimated from cyclic voltammograms at low scan rate (10 mV s −1 , lower scan rates did not yield any noticeable redox peaks), according to the following equation: ΓOs = Q(nFA) −1 , where Q is the charge for the redox conversion of the polymer-bound Os-complexes, n is the number of electrons transferred, F is the Faraday constant, and A is the geometric electrode area.
A Langmuir-Blodgett (LB) trough (5.5 cm × 54.0 cm, KSV Instruments) was used for preparation and transfer of PSI monolayers onto the electrode substrates, as it has been described in detail before. [2] Briefly, a software enabled to control two symmetric barriers for monolayer formation at constant compressing speed and subsequent monolayer transfer at constant surface pressure using a Pt Wilhelmy plate to determine the surface pressure. 5 mM phosphate buffer pH 7.0 was used as subphase, on top of which diluted solutions of PSI trimers, monomers or mixtures were deposited. The PSI solution was spread gently at the air/water interface of the subphase to avoid falling of material into the bulk of the solution. LB transfer was performed at a rate of 5 mm min −1 at 20 °C. For more experimental details and a description of the setup used, the reader is referred to ref. [2] The amount of PSI over the electrode surface was estimated by chlorophyll detection via methanol extraction. For this, three different electrodes modified with PSI LB films (modified surface area: 3.6 cm 2 ) were incubated with 5 mL methanol in a shaker for 60 min. After subsequent evaporation of the solvent, the extracted chlorophyll for each electrode was resuspended in 30 µL methanol. After sedimentation at 15,000 g, an aliquot of 10 µL was loaded on standard treated MST capillaries (NanoTemper Technologies, Inc.) and chlorophyll fluorescence was measured via Monolith NT.155 (NanoTemper Technologies, Inc.), together with an internal chlorophyll standard, at an excitation power of 1% between 605 nm and 640 nm. The initial fluorescence was detected via a Nano RED detector in a range between 670 nm and 730 nm and used for chlorophyll loading calculations.

Electrochemical Measurements
A conventional three-electrode setup was used, consisting of the PSI modified substrate as working electrode (with a surface area of 0.246 cm 2 ), a Ag/AgCl/3 M KCl reference electrode, and a Pt mesh as counter electrode. Cyclic voltammetry and photochronoamperometric measurements were performed using a PGU-BI 100 potentiostat (IPS Jaissle). For illumination of the sample a He-Xe lamp (LC8 type 03, Hamamatsu Photonics) was used, integrating a red foil filter (No. 164 LEE Filters, λ > 600 nm) and with an incident power of 51 mW cm −2 , unless stated otherwise. Electrochemical impedance spectroscopy measurements were performed using an Autolab PGSTAT302N potentiostat (Metrohm-Autolab) in a 0.1 M KCl solution containing equimolar concentrations of [Fe(CN)6] 4− and [Fe(CN)6] 3− (5 mM each). The experiments were conducted at the equilibrium potential of the redox couple (DC potential of 236 mV vs. Ag/AgCl/3 M KCl) superimposed with an AC perturbation of 10 mVrms amplitude in a frequency range comprised between 100 kHz and 100 mHz.

Gas Chromatography Detection
H2 was detected using a gas chromatograph (GC, multiple gas analyzer #1, MG#1, SRI Instruments) equipped with a 3 meters HayeSep D column at 90 °C and a thermal conductivity detector (TCD). The detection with the modified sample was performed using a small-volume electrochemical cell (total volume: 180 µL, headspace: 50 µL), using N2 to generate an inert atmosphere. After 36 minutes of a constant applied potential of 160 mV vs. SHE, an aliquot of 25 µL of the headspace was collected and manually on-column injected into the GC. For correlation of the retention time, a H2 standard with a concentration of 800 ppm was injected by quadruplicate.

Detergent exchange
Since LDAO in the monomer sample was suspected to interfere with the formation of PSI monolayers, a detergent exchange to DDM was carried out via ion exchange chromatography (IEC). 0.5 mg of isolated monomeric PSI were mixed with 30-fold excess of exchange buffer (20 mM HEPES pH 7.5, 10 mM MgCl2. 10 mM CaCl2, 500 mM mannitol, 1% (w/v) DDM) and incubated for 10 min before they were loaded on an IEC column (Uno Q6R, Bio Rad, Germany) integrated in the AKTA purifier system, previously described. The column was previously equilibrated (20 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM CaCl2, 500 mM mannitol, 0.03% (w/v) DDM) and PSI was eluted via 130 mM MgSO4 over 4 column volumes. PSI-containing fractions were washed via centrifuge concentrators (MWCO 100 kDa, Millipore ® , Merck, USA) with equilibration buffer, concentrated to 2 mg PSI x mL −1 and flashfrozen in liquid N2.

Synthesis of BPEI-[CoCp2]
The synthesis of the cobaltocene-functionalized branched polyethyleneimine BPEI-[CoCp2] (Scheme S1) was conducted as follows: 20 mg of branched polyethyleneimine (BPEI, 50 wt% in H2O) were dried under reduced pressure at room temperature overnight. Subsequently, the dried BPEI was dissolved in 1 mL of dry DMSO under Aratmosphere and 12 µL of Ar-saturated triethylamine (Et3N) were added and the solution was stirred for 5 min. After this, 12 mg (0.025 mmol) of 1-(2,5-dioxopyrrolidinylcarboxy)-cobaltoceniumhexafluorophosphate ([CoCp2]-NHS, MCAT GmbH) were dissolved in 0.5 mL of dry DMSO and slowly added to the BPEI solution resulting in the solution to turn yellow. Subsequently, the solution was stirred for 5 h at room temperature. After the reaction was finished, 10 mL of diethyl ether were added to the solution, resulting in precipitation of a yellow oil. The product was washed with 2 × 10 mL of diethyl ether, 10 mL of dichloromethane, and 10 mL of methanol. The product was then dried at room temperature under reduced pressure. The dried product was dissolved in 2 mL of water. Subsequently, the polymer solution was subjected to dialysis by centrifugation against 0.1 M KCl over 5 kDa molecular weight cut-off membrane filters (8 × 20 min at 8,000 rpm) to replace the hydrophobic PF6 − counterions with hydrophilic Cl − counterions. Finally, the polymer was washed with H2O by centrifugation over 5 kDa membrane filters (3 × 20 min at 8,000 rpm) to remove excess KCl, dissolved in 2 mL of H2O obtaining a concentration of 13.5 mg mL −1 , and stored at 4 °C.